When determining the microbiological quality of biological materials, e.g. determining the number of bacteria in meat meant for human consumption, various attempts have been made to utilize bioluminescence reactions, in particular the so-called "firefly"-reaction or luciferin/luciferase reaction. In this reaction, adenosine triphosphate (ATP) reacts with luciferin in the presence of the enzyme luciferase while generating light, and this method is a rapid and sensitive method for determining ATP in a sample. Since ATP is present in practically all living cells, the method can be used for detecting the presence of somatic (eucaryotic), bacterial and fungal cells in a sample. When, as in the present case, the purpose particularly is to determine the number of bacteria, it becomes important to ensure that non-bacterial cells in the sample, in particular somatic cells (such as tissue cells from a meat sample) do not interfere with the measurement by contributing to the bioluminescence reaction with their own ATP. In other words, it becomes important to ensure that only ATP from bacterial cells is subjected to the bioluminescence reaction whereas it should be prevented that ATP from somatic cells takes part in the reaction.
One way of achieving this would be to selectively lyze the somatic cells without disturbing the microbial cells, but methods used so far invariably describe the use of some kind of active lyzing agent which commonly is some kind of surfactant. Thus, U.S. Pat. No. 4,303,752 describes the selective removal of ATP from somatic cells in a mixture of somatic and microbial cells by treating the sample with a non-ionic surfactant, e.g. an ethoxylated alkyl phenol. However, it is also known that a number of non-ionic surfactants can cause inhibition of the luciferase enzyme in the subsequent bioluminescence assay which means that any remains of non-ionic surfactant that has not been removed will significantly reduce the light output and consequently reduce the sensitivity of the assay.
With respect to the subsequent bioluminescence assay, previous commercially available methods for carrying out this assay have employed glass containers (cuvettes), in which both rupture of the somatic cells, inactivation of the released somatic ATP, release of bacterial ATP, and reaction with luciferin/luciferase takes place in the same container. Such a system has a number of drawbacks. Firstly, since the somatic ATP cannot be removed physically from the medium, it is necessary to use an enzyme (somase) to render the somatic ATP inactive, which in turn necessitates the subsequent inactivation of the enzyme prior to the release of the bacterial ATP. Secondly, the presense of cell fragments and remains in the medium reduces the sensitivity of the luminescent reaction because of scattering of the light emitted during the reaction. Consequently, this method is not well suited to determining the low concentrations of bacteria encountered within the food quality control field, quite apart from the extra several inactivation steps necessary.
Other suggested methods which, however, have not resulted in commercially available instruments, have employed having the now purified bacterial material present on the surface of a substrate such as a filter surface and lowering a container part onto the substrate to form a closed measuring chamber, the container part comprising means for detecting light such as a photomultiplier. However, the fact that one of the walls of the measuring chamber is formed by the filter surface, on which the bacteria to be counted are situated, makes it difficult to ensure that the measuring chamber is completely light-proof; the porous and/or translucent quality of most filtering materials will cause difficulties when attempting to provide a completely light-proof seal between the container part and the surface of the filter material.